Comparative non-cholinergic neurotoxic effects of paraoxon and diisopropyl fluorophosphate (DFP) on human neuroblastoma and astrocytoma cell lines.
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The objective of this study was to evaluate the comparative non-cholinergic neurotoxic effects of paraoxon, which is acutely neurotoxic, and diisopropyl fluorophosphate (DFP), which induces OPIDN, in the human neuroblastoma SY5Y and the human astrocytoma cell line CCF-STTG1. SY5Y cells have been studied extensively as a model for OP-induced neurotoxicity, but CCF cells have not previously been studied. We conducted a preliminary human gene array assay of OP-treated SY5Y cells in order to assess at the gene level whether these cells can distinguish between OP compounds that do and do not cause OPIDN. Paraoxon and DFP induced dramatically different profiles of gene expression. Two genes were upregulated and 13 downregulated by at least 2-fold in paraoxon-treated cells. In contrast, one gene was upregulated by DFP and none was downregulated at the 2-fold threshold. This finding is consistent with current and previous observations that SY5Y cells can distinguish between OPs that do or do not induce OPIDN. We also examined gene array results for possible novel target proteins or metabolic pathways for OP neurotoxicity. Protein levels of glucose regulated protein 78 (GRP78) revealed that paraoxon exposure at 3 microM for 24 h significantly reduced GRP78 levels by 30% in neuroblastoma cells, whereas DFP treatment had no effect. In comparison with SY5Y neuroblastoma cells, paraoxon and DFP (3 microM for 24 h) each significantly increased GRP78 levels by 23-24% in CCF astrocytoma cells. As we have previously evaluated intracellular changes in Ca(2+) levels in SY5Y cells, we investigated the effects of paraoxon and DFP on cellular Ca(2+) homeostasis in CCF by studying cytosolic and mitochondrial basal calcium levels. A significant decrease in the ratio of mitochondrial to cytosolic Ca(2+) fluorescence was detected in CCF cultures treated for either 1 or 3 days with 1, 3, 10, or 30 microM paraoxon. In contrast, treatment with DFP for 1 day had no significant effect on the ratio of mitochondrial to cytosolic Ca(2+) fluorescence; after 3 days treatment, only 30 microM decreased the ratio. These results are consistent with the finding that paraoxon induced a greater decrease than did DFP of intracellular esterase activity in CCF cells. The changes seen in the ratio of mitochondrial to cytosolic Ca(2+) represent a good indicator of the degree of injury induced by each chemical tested. This work further develops in vitro models that distinguish between compounds that cause OPIDN and those that induce acute neurotoxicity only. The study also exposes additional OP-induced toxicities that may be obscured in vivo.
